1. Field of the Invention
The present invention relates to a method using a cell penetrating peptide (Pep-1) for labeling and delivering mitochondria separated from normal cells to replace dysfunctional mitochondria in deficient cells.
2. Description of the Prior Art
The necessity and demand for mitochondria transplantation is high because mitochondrial defects are involved in many diseases with unknown reasons and no cure is currently available. Majority of the DNA mutations found in organelles (mitochondria) in cytoplasm are inherited genetic diseases; yet, some are caused by gene mutations. The defects are usually passed from mothers to their children because each egg has thousands of mitochondria, and upon fertilization, the mitochondria of sperms were left outside of the zygote; hence, the mitochondrial DNA in the zygote all came from the mother. The incidence of mitochondrial defects and the distribution of these rare diseases caused by mitochondrial DNA, are random since each cell contains thousands of mitochondria, and each mitochondria has 2˜10 mitochondrial DNA; which mitochondrial DNA has defects is uncertain. In Taiwan, around 50 mitochondrial defects—related diseases were reported up to date, and these diseases are categorized as rare diseases; furthermore, roughly 300˜400 domestic families in Taiwan are found to have mitochondrial DNA defects with a defect rate around 1/10,000.
Mitochondrial defects are widely involved in numerous diseases and malignancies with ever-changing clinical symptoms. For the age of the onset of the diseases, from newborn babies to adults, some symptoms are persistent and apparent (e.g. developmental retardation and seizure, etc); On the other hand, some are non-specific symptoms such as migraine and short stature, etc., and only become apparent when in poor physical conditions. Different organs may have various degrees of lesions, for instance, headache, seizures, dementia, cortical blindness, partial paralysis, mental retardation, motor development retardation, and brain stem abnormalities, etc. found in the brain; different levels of muscular abnormalities; myocardial hypertrophy, atrioventricular conduction abnormalities found in the heart; eyelids dropping, outer eye muscle paralysis, optic nerve degeneration, and retinopathy found in eyes; kidney tubular function abnormalities; liver function abnormalities; vomiting, diarrhea, pseudo intestinal obstruction found in the GI tract; diabetes caused by defects in the pancreas, and bone marrow malfunction. Other symptoms include deafness, short stature, injury to the peripheral nervous system, and subcutaneous lipoma, etc. In addition, a number of symptoms or diseases of aging are related to mitochondrial defects, including atherosclerosis, stroke, Parkinson's disease, Alzheimer's disease, and cancers.
Up to date, there are no cures available for mitochondria-related diseases and the most common treatment is vitamins or pyruvate. Yet, several therapies have been developed along with the expanding medical knowledge of mitochondria-related diseases, and these treatments can assess individual conditions and improve the symptoms. Theoretically, maternally inherited mitochondrial genetic defects may be prevented by transplantation using microinjection of single embryo mitochondria; however, for mutations in mitochondria caused by external factors such as oxidation stress or poor quality of the mitochondria, the present invention can provide a method that can significantly improve the efficiency of transplantation of mitochondria into a large number of cells at one time.
Mitochondria are the main energy source in eukaryotic cells; moreover, cell growth, cell differentiation, and even cell death are all regulated by mitochondria. Literatures indicate that cell fusion of stem cells and mitochondria-malfunctioned cells permit the delivery of mitochondria between cells, and consequently facilitate the repairing process of the damaged cells and restore the normal energy metabolism pathway. Therefore, positive or passive transplantation of “cell mitochondrion” has become an effective cell therapy method. Nonetheless, application of the technology remains limited due to different transplantation efficiency and cell properties. For example, only one cell can be treated using microinjection, and as a result, the efficiency of treatment is poor or the delivered mitochondria in treated cells are not transferred by fusion with other cells. Hence, no effective method that can positively deliver mitochondria into cells is available at present.
The idea of mitochondria transplantation was brought up in foreign literatures in 1998, and recent studies have also indicated that using transgenic technology to transplant mitochondria into embryos can enhance embryo development. To evaluate the effects of mitochondria transplantation on embryo development, mitochondria were separated from mouse liver cells and microinjected into the zygotes at 2PN stage collected from either young or old mice. In vitro culture of the above zygotes showed that in young mice, the percentage of the embryos developed to the blastocyst stage were significantly higher in the experimental group (37.65%) than in the control group (20.91%); however, the hatching rate was not affected (experimental group is 1.76% and control group is 1.82%). Even for the zygotes collected from older mice (about 20 weeks old), development of these embryos in the control group was also notably improved (experimental group vs. control group is 54.35% vs. 18.92% and 43.48% vs. 8.11% for embryos at morula stage and blastocyst stage, respectively). Due to ethical issues and potential risks including mitochondria heterogeneity and effects on cell activity, etc. relevant studies focused on mitochondrial transplantation in humans are not currently available. Additionally, whether the mode of transplantation is suitable for use in other cell types or other interventions are applicable requires further investigation.
Currently, mitochondrial transgenic technologies include microinjection, cell infusion, tRNA transgenic system, and peptide-mediated mitochondrial delivery system (PMD). Their respective principles, treating objects, interventional methods, and advantages/disadvantages are shown in Table 1. Among which, the peptide-mediated mitochondrial delivery system (PMD) can process a large number of cells at one time, and the number of delivered mitochondria can be regulated accordingly; moreover, the process is simple and time efficient, and no advanced techniques are required.
TABLE 1Comparison of mitochondrial transgenic technologies.MethodPeptide-mediatedmitochondrialtRNA transgenicdelivery systemMicroinjectionCell fusionsystemPrincipleDeliversDeliversPassive deliveryDeliversmitochondriamitochondriavia mitochondrialmitochondriaobtained andseparated frominteractionseparated fromseparated fromhealthy cellsinduced by stemLeishmaniahealthy cells.cell fusionRNA importcomplex (RIC)TreatingMERRF patient-Mouse oocytesHuman A549Human MERRFobjectsderivedρ° cells treatedpatient-derivedmitochondria fusionwith EthidiumLB64 cells andcell line (B2 clone)Bromide tomitochondrialand human B143 ρ°inhibitfusion cell linecells treated withmitochondriaKSS (FLP32.39)Ethidium Bromideto inhibitmitochondriaInterventionalCell penetratingMicroinjectionCo-cultureLiposomemethodspeptide (Pep-1)coatingDisadvantages1. Delivery1. One cell at a1. Translocation1. Tediousefficiency istime.of the healthyprocedures.below 100%.2. Timemitochondria2. Advanced2. Averageconsuming.into thetechnologynumbers of3. Requiresdamaged cellsrequired.mitochondriaadvancedcannot be3. High cost anddelivered into thetechnology.controlled.pre-treatmentcells cannot be2. Not suitableis time -controlledfor all cellconsumingaccurately.types, because4. tRNA cannotcell fusionbe stored °may not occurin certain cellseven after co-culture.Advantages1. Large number ofSingle cell1. No activeDirect regulationcells can betransgenic rateinterventions.of tRNAtreated at oneis up to 100%.2. The simplestproteins, and cantime.and leastcompletely2. The process ofexpensiverepair themitochondrialmethod.defects causeddelivery can beby mutations inregulatedmitochondrialaccordingly.DNA.3. No advancedtechnologyrequired.
Cell penetrating peptide (Pep-1) belongs to the cell penetrating peptide families and has the sequence as shown in SEQ ID NO: 1. Pep-1 consists of 3 domains: hydrophobic domain (KETWWETWWTEW), hydrophilic domain (contains numerous lysine (K), KKKRKV), and spacer (SQP). The peptide contains both hydrophobic and hydrophilic ends, and is an amphipathic peptide. These types of peptides are usually used as drug and enzyme carriers, and they deliver their cargos by forming the particles using hydrophobic ends which cannot dissolve in water. Drugs or enzymes are then incorporated in a self-assembling reaction during the embedding process where the positive charge carried by hydrophilic domain binds to the negative charge on the cell membrane. Meanwhile, the hydrophobic domain integrates into the cell phospholipid bilayer and transports proteins into the cell. The advantage of this method is that prior chemical treatment of the target protein is not required, and the mechanism of cell entry is positive delivery, which is independent of endocytosis. Thus, the target protein will not be directly catalyzed in lysosomes, and increase cytoplasm conservation. Previous studies have also shown that Pep-1 has no cell toxicity, and its delivery will not affect ligands binding to their receptors on the cell surface.
Pep-1 peptide used in the present invention has been applied in delivery of drugs and particles; nonetheless, delivery of mitochondria using Pep-1 is a new technology developed by the present invention. In addition, by combination of the advanced mitochondria separation method, we further developed the peptide-mediated mitochondrial delivery system (PMD) which has the following advantages: the procedures are easy to follow and time efficient, one labeling process can treat numerous cells, the desired mitochondria quantity (μg) can be controlled accordingly, no cell toxicity under appropriate transplantation conditions, and transplantation efficiency can reach up to 80%. The mitochondria delivered using this system will move to the original mitochondrial sites in the cells, and will not be catalyzed in the lysosomes; hence, the therapeutic effects can last at least one week.
The present invention relates to a method using a cell penetrating peptide (Pep-1) for labeling and delivering mitochondria separated from healthy cells to replace damaged mitochondria, and can be used to treat cell mitochondrial degeneration and related diseases.
In summary, after years of painstaking research, the inventor(s) of the present invention successfully developed the novel peptide-mediated mitochondrial delivery system (PMD) and demonstrated various applications of the newly developed system.